Degradation of epothilones

ABSTRACT

According to one embodiment the invention concerns a process for a degradation of an epothilone C or a epothilone D, wherein an epothilone C or epothilone D is subjected to an olefin metathesis in the presence of ethylene and subsequently an optional ester hydrolysis.

Epothilones of type C and type D belong to the art and are especiallycharacterized by a C═C double bond at positions 12 and 13 and a hydrogenatom at position 12 (type C) or an alkyl group-(type D).

According to one embodiment the invention concerns a process for adegradation of an epothilone C or an epothilone D, wherein an epothiloneC or an epothilone D is subjected to an olefin metathesis in thepresence of ethylene and subsequently an optional ester hydrolysis(scheme I).

According to the invention the epothilone C or D can be a fermentationproduct.

According to another embodiment the invention concerns a process for theproduction of an epothilone of formula 9

wherein an epothilone of formula 2a (schemes I and II) is converted intocompound of formula 3a (scheme II), the compound of formula 3a isreacted with a compound of formula 6 (which has been formed by reactinga compound of formula 4 with a compound of formula 5; scheme II) to givea compound of formula 7 by esterification (scheme II), the compound offormula 7 is reacted in the presence of a Grubbs catalyst to give acompound of formula 8a by deprotection (scheme II), the compound offormula 8a is converted into a compound of formula 8b by deprotection(scheme II), and compound of formula 8b is converted to a compound offormula 9 by epoxidation (scheme II).

Alternatively to the reaction sequence depicted in scheme I syntheticintermediates of type 3 may be obtained according to scheme III by

-   1) cleavage of the lactone of epothilone C or D with e.g. pig liver    esterase (PLE) or, after protection of the 3,7-hydroxyl groups, with    aqueous base to give 10 (this conversion is described in U.S. patent    application Ser. No. 09/811,808, Mar. 19, 2001 by BMS/GBF),-   2) optionally esterification with diazomethane and optionally    protection of the 3,7-dihydroxyl groups to give 11,-   3) olefin metathesis with an excess of an olefin, e.g. ethylene and    a ruthenium or molybdenum metathesis catalyst and optionally    protection of the 3,7-dihydroxyl groups to give 3b.

EXPERIMENTAL PART 12,13-seco-Epothilone C (2a)

450 mg of epothilone C (1) (0.95 mmol) were dissolved in 250 mL ofdichloromethane, saturated with ethylene and after addition of 60 mg ofGrubb's catalyst (PhCHRuCl₂[P(Cy)₃]₂ stirred for 24 hours. Afteraddition of further 60 mg of catalyst and stirring for 24 hours the darksolution was evaporated to dryness and the residue purified bychromatography on silica with the solvent systemhexanes/tert.-butylmethylester/methanol 80:20:1. The first fractioncontained 360 mg (75%) of 2a, the second 100 mg (22%) of recoveredstarting material 1.

2a: ¹H-NMR (CDCl₃), 300 MHz): δ=6.95 (s, 19-H), 6.02 (s, 17-H),5.89–5.64 (m, 12-H, 13-H), 5.16–4.89 (m, 12a-H₂, 13a-H₂), 5.37 (t, J=7Hz, 15-H), 4.24 (ddd, J=10, 3, 3.5 Hz, 3-H), 3.36 (s, OH), 3.34 (d, J=8Hz, 7-H), 3.25 (dq, J=1.5, 7 Hz, 8-H), 3.21 (d, J=3.8 Hz, OH), 2.70 (s,21-H₃), 2.52–2.32 (m, 2-H₂, 14-H₂), 2.07 (d, J=1.5 Hz, 16-Me), 2.05–1.95(m, 11-H₂), 1.8–1.1 (m, 6-H, 8-H, 9-H₂, 10-H₂), 1.18 (S, 4-Me), 1.10 (s,4-Me), 1.04 (d, J=7 Hz, 6-Me), 0.83 (d, J=7 Hz, 8-Me).

ESI-MS (pos ions) m/z=506 [M+H⁺], CI-MS (NH₃ pos. ions) m/z=506 [M+H⁺](22%), 380 (100%).

3,7-Di-[tert-buthyldimethyl-silyloxy]-4,4,6,8-tetramethyl-5-oxo-12-tridecenoicacid (3a)

To 330 mg (0.65 mmol) of 12,13-seco-epothilone C (2a) dissolved in 10 mLof THF were added with stirring 0.6 mL of NEt₃ and 0.6 mL oftert-butyldimethylsilyltriflate. After one hour the solvent wasevaporated in vacuo. The residue was dissolved in 10 mL of THF, 70 mg ofLiOH dissolved in 0.5 mL of water were added and the mixture stirred for16 hours. The solvents were evaporated and the residue distributedbetween phosphate buffer of pH 5 and ethyl acetate. The organic layerwas dried with MgSO₄ and evaporated to dryness. Preparative HPLC onRP-18 with the solvent system methanol/20 mmol ammonium acetate bufferpH 7 gave 235 mg (67%) of 3a as colorless viscous oil.

Analytical HPLC on Nucleosil RP-18 (260×5 mm) solvent system methanol/20mmol ammonium acetate buffer pH 7, 1 mL/min, light scattering detector:R_(t)=5.5 min.

¹H-NMR (CDCl₃, 300 MHz): δ=5.78 (m, 12-H), 4.99, 4.92 (m, 13-H₂), 4.39(dd, J=6.3, 3.4 Hz, 3-H), 3.79 (dd, J=7.2, 2.0 Hz, 7-H), 3.12 (dq, J=7.0Hz, 8-H), 2.49 (dd, J=16.5, 3.5 Hz, 2-H_(a)), 2.32 (dd, J=16.5, 6.2 Hz,2-H_(b)), 1.5–1.0 (m, 6-H, 8-H, 9-H₂, 10-H₂, 11-H₂), 1.2 (s, 4-Me), 1.07(s, 4-Me), 1.04 (d, J=6.9 Hz, 6-Me), 0.91 (d, J=7.0 Hz, 8-Me), 0.89 (s,tBuSi), 0.88 (s, tBuSi), 0.09 (s, MeSi), 0.06 (s, MeSi), 0.05 (s, 2MeSi).

ESI-MS (neg. ions) m/z=541 (M−H).

4-Bromo-2-methyl-thiazole (4)

1 g (2.05 mmol) 2,4-Dibromothiazole was dissolved in 25 mL anhydrousether and the resulting solution was stirred under N₂ atmosphere at −78°C. To the solution was added n-BuLi (1.1 equivalent, 4.52 mmol, 2.82 mLof 1.6 M solution in hexane) and the stirring was continued for 1 h. Tothe reaction mixture was then added dropwise a solution ofdimethylsulfate 1.16 mL (12.34 mmol) in 1 mL ether. After stirring for 4h at −78° C. the reaction mixture was allowed to warm to roomtemperature and stirred for 14 h. The reaction mixture was diluted witha saturated solution of NaHCO₃ (10 mL). The aqueous layer was extractedwith ether and the combined organic extracts were washed with a brineand dried over MgSO₄. Concentration under vacuum, and flash columnchromatography (silica gel, 10:1 petroleum ether/ethyl acetate), yielded0.52 g (70.6%) a yellow oil.

IR (KBr) 3122, 2923, 1485, 1441, 1252, 1178, 1085887, 834 cm⁻¹.

¹H-NMR (CDCl₃, 400 MHz): δ=7.02 (s, 1H), 2.71 (s, 3H).

¹³C-NMR (CDCl₃, 100.6 MHz): δ=167.31, 124.18, 116.11, 19.40. EI-MS (70eV): m/z (%): 179 (93) [M+2H]⁺, 177 (100) [M+H]⁺, 169 (30), 164 (20),159 (15).

HRMS (EI): calcd for C₄H₄BrNS 176.9251, found 176.9248

1-(2-methyl-thiazol-4-yl)-hex-5-en-1-yn-3-ol (6)

480 mg (2.68 mmol) 4-Bromo-2-methyl-thiazole (4) in 4 mL Et₃N was addedto 131 mg (0.187 mmol) PdCl₂(PPh₃)₂ and the suspension was stirred 15minutes under N₂ atmosphere at room temperature then 117 mg (0.614 mmol)CuI was added under N₂ atmosphere followed by dropwise addition of 283mg alcohol 5 (A. B. Smith, III et al. JACS 120, 3935–3948 (1998)) in 1mL Et₃N. The mixture was stirred for 15 minutes at room temperature andheated to 80° C. for 6 h. Concentration under vacuum, and flash columnchromatography (silica gel, 3:2 petroleum ether/ethyl acetate), yielded0.29 g (56%) a yellow oil. [α]=−29.1 (c=1 in chloroform)

IR (KBr): 3386, 3142, 2924, 1641, 1501, 1435, 1286, 1194, 1041, 993, 918cm⁻¹.

¹H-NMR (CDCl₃, 400 MHz): δ=7.26 (s, 1H), 5.98–5.88 (m, 1H), 5.23–5.16(m, 2H), 4.62 (dd, J=11.9, 5.8 Hz, 1H), 2.68 (3H, S), 2.58–2.54 (2H, m),2.39 (d J=6.1 Hz, 1H, OH)

¹³C-NMR (CDCl3, 75.5 MHz): δ=165.77, 136.20, 133.09, 122.48, 118.85,89.53, 79.04, 61.84, 41.87, 19.10.

DCI-MS (NH₃): 211[M+NH₄ ⁺], 194[M+H⁺].

(1S)-1-[(2-Methyl-thiazole-4-yl)-1-ethynyl]-3-butenyl (3S, 6R, 7S,8S)-3,7-di-[tert-butyldimethylsiloxy]-4,4,6,8-tetramethyl-5-oxo-12-tridecenoate(7)

99 mg (0.478 mmol)DCC was added at 0° C. to a solution of acid 200mg(0.368 mmol), alcohol 79 mg (0.405 mmol) and 12 mg (0.09 mmol) DMAP in10 mL CH₂Cl₂. The mixture was stirred for 15 min at 0° C. and for 16 hat room temperature. Concentration under vacuum, and flash columnchromatography (silica gel, 10:1 petroleum ether/ethyl acetate), yielded240 mg (91%) a yellow oil.

[α]=−45.8 (c=1 in CH₂Cl₂)

IR (KBr): 2929, 2856, 1742, 1697, 1641, 1472, 1253, 989 cm⁻¹.

¹H-NMR (CDCl₃, 400 MHz): δ=7.28 (s, 1H, thiazole H-5), 5.91–5.73 (m, 2H,H-12, H-3′),

5.58 (t, J=6.1 Hz, 1H, H-1′), 5.20–4.90 (m, 4H, H-13, H-4′), 4.38 (dd,J=6.3, 3.3 Hz, 1H, H-3), 3.74 (dd, J=6.8, 2.2 Hz, 1H, H-7), 3.11 (dq,J=6.8, 6.8 Hz, 1H, H-6), 2.67 (s, 3H, thiazole CH₃), 2.60 (t, J=6.6 Hz,1H, H-2), 2.55 (dd, J=16.7, 3.5 Hz, 1H, H-2′), 2.29 (dd, J=17.0, 63 Hz,1H, H-2′), 2.05–1.95 (m, 2H, H-11), 1.47–1.29 (m, 3H,) 1.17–1.08(m, 2H)(H-8, H-9, H-10), 1.21 (s, 3H, H-22), 1.05 (s, 3H, H-23), 1.03 (d, J=6.6Hz, 3H, C6-CH₃), 0.89 (d, J=6.6 Hz, 3H, C8-CH₃), 0.88, 0.87(2s, 2x9H,OSiC(CH₃)₃), 0.089 (s, 3H, OSi(CH₃)₂), 0.032, 0.028, 0.024 (3s, 3x3H,OSi (CH₃)₂).

¹³C-NMR (CDCl₃, 100.6 MHz): 217.63, 170.84, 165.55, 138.97, 136.08,132.23, 123.22, 118.91, 114.41, 85.67, 79.97, 73.76, 63.77, 53.38,45.23, 40.20, 39.09, 38.87, 34.35, 34.00, 30.48, 27.11, 26.26, 26.07,25.66, 24.97, 23.44, 19.89, 18.55, 17.66, 15.52, −3.61, −3.74, −4.20,−4.59

DCI-MS (NH₃): 735[M+NH₄ ⁺], 718[M+H⁺].

HRMS (DCI): calcd for C₃₉H₇₀N₂O₅SSi₂ 735.4622, found 735.4675.

(4S, 7R, 8S, 9S,16S)-4,8-Di-tert-butyldimethylsilyloxy-5,5,7,9-tetramethyl-1-6-(2-(2-methyl-1,3-thiazol-4-yl)-1-ethynyl)-1-oxa-13-cyclohexadecen-2,6-dione,mixture of Z and E isomeres (8a)

To a solution of 190 mg (0.264 mmol) diene 7 in 66 mL CH₂Cl₂ was added44 mg (0.053 mmol)

bis(tricyclohexylphosphine)benzylideneruthenium dichloride and thereaction mixture was stirred for 48 h at room temperature. Concentrationunder vacuum, and flash column chromatography (silica gel, 10:1petroleum ether/ethyl acetate), yielded 95 mg (52%) of a yellow oil.

(4S, 7R, 8S, 9S,16S)-4,8-Dihydroxy-tert-5,5,7,9-tetra-methyl-1-6-[2-(2-methyl-1,3-thiazol-4-yl)-1-ethynyl)-1-oxa-13-cyclohexadecen-2,6-dione(8b), mixture of cis and trans isomere

A solution of 95 mg (0.137 mmol) lactone X in 12 mL CH₂Cl₂ at −20° C.was treated with 2 mL trifluoroacetic acid, and the mixture was stirredfor 2 h at 0° C. After concentration under vacuum, the residue wasdiluted with EtOAC, washed with saturated NaHCO₃ solution and dried overMgSO₄. Concentration under vacuum, and separation by HPLC (80:20:3hexane/t-BuOMe/MeOH), yielded 27 mg (42%) of the cis-hydroxy lactone 8band 27 mg (42%) of the corresponding trans isomer.

[α]=−123 (c=1 in CH₂Cl₂)

¹H-NMR (CDCl₃, 400 MHz): δ=7.30 (s, 1H, H-19), 5.65 (dd,J=9.1, 2.9 Hz,1H, H-15), 5.55–5.41 (m, 2H, H-12, H-13), 4.20 (dd,J=10.8, 2.7 Hz, 1H,H-3), 3.67–3.65 (m, 1H, H-7), 3.12 (dq, J=6.6, 2.0 Hz, 1H, H-6),2.88–2.77 (m, 1H, H-14), 2.70 (s, 3H, H-21), 2.51 (dd, J=15.0 Hz, 10.9Hz, 1H, H-2), 2.27 (dd,J=15.2, 2.8 Hz, 1H, H-2), 2.18–2.00 (m, 2H, H-11,H-14), 1.71–1.58 (m, 3H, H-8, H-9, H-10), 1.32 (s, 3H, H-22), 1.30–1.19(3H, H-8, H-9, H-10), 1.18 (d, J=6.7 Hz, 3H, H-24), 1.07 (s, 3H, H-23),0.98 (d, J=6.9 Hz, 3H, H-25)

¹³C-NMR (CDCl3, 75.5 MHz): δ=220.81, 169.96, 164.44, 134.16, 134.27,123.75, 123.00, 86.13, 80.00, 74.38, 72.03, 64.11, 53.31, 41.74, 39.37,38.71, 32.87, 32.37, 27.63, 27.47, 22.69, 19.18, 18.37, 15.46, 13.70.

16,17-Didehydro-16-desmethyl-epothilone A (9)

To a solution of 27 mg (0.058) of lactone (8b) 4 mL CH₂Cl₂ was addeddropwise at −20° C. a solution of dimethyl dioxirane in acetone (2equiv). Stirring was continued for 2 h at −20° C. Concentration undervacuum, and separation by HPLC (80:20:3 hexane/t-BuOMe/MeOH), yielded 17mg (60%) of α-epoxide 9 and 9 mg (32%) of β-epoxide.

α-epoxide

[α]=−34 (c=1 in CH₂Cl₂)

IR (KBr): 3453, 2958, 2850, 1744, 1690, 1500, 1467, 1376, 1290, 1261,1147, 979, 775 cm⁻¹.

¹³C-NMR (CDCl₃, 100.6 MHz): 220.55, 170.19, 166.12, 135.50, 123.28,85.00, 80.56, 75.12, 73.59, 62.71, 57.17, 53.75, 52.67, 43.68, 38.69,35.96, 32.67, 29.72, 26.56, 23.63, 21.12, 20.48, 19.16, 17.06, 14.46

EI-MS (70 eV): m/z (%): 477(27) [M+H]⁺, 421 (14), 389 (19), 378 (100),364 (28), 346 (27), 328 (15).

β-epoxide

¹³C-NMR (CDCl₃, 75.5 MHz): δ=221.38, 170.03, 166.05, 135.70, 123.28,85.13, 80.48, 73.24, 73.11, 62.24, 57.14, 55.31, 52.28, 42.89, 38.98,37.53, 32.40, 31.82, 27.60, 27.01, 23.45, 20.62, 20.36, 16.38, 13.49.

1. A process for a degradation of an epothilone C or an epothilone D,comprising subjecting an epothilone compound having the formula,

wherein P is a protecting group, and R is hydrogen (epothilone C) ormethyl (epothilone D), to an olefin metathesis in the presence ofethylene and catalyst to produce a compound having the formula 2,

and subsequently performing an ester hydrolysis of the compound offormula 2 in the presence of base or hydrolytic enzymes to produce acompound having the formula 3,

wherein M is an alkali metal or hydrogen.
 2. The process according toclaim 1, wherein the epothilone C or D is a fermentation product.
 3. Theprocess according to claim 1 wherein P is trialkylsilyl ortert-butyl-dimethylsilyl.
 4. The process according to claim 1 whereinthe catalyst is an Ru and/or Mo metathesis catalyst.
 5. The processaccording to claim 1 wherein the enzyme is pig liver sterase.